We provide here an exhaustive overview of the glutathione (GSH) peroxidase (Gpx) family of poplar (Populus trichocarpa). Although these proteins were initially defined as GSH dependent, in fact they use only reduced thioredoxin (Trx) for their regeneration and do not react with GSH or glutaredoxin, constituting a fifth class of peroxiredoxins. The two chloroplastic Gpxs display a marked selectivity toward their electron donors, being exclusively specific for Trxs of the y type for their reduction. In contrast, poplar Gpxs are much less specific with regard to their electron-accepting substrates, reducing hydrogen peroxide and more complex hydroperoxides equally well. Site-directed mutagenesis indicates that the catalytic mechanism and the Trx-mediated recycling process involve only two (cysteine [Cys]-107 and Cys-155) of the three conserved Cys, which form a disulfide bridge with an oxidation-redox midpoint potential of 2295 mV. The reduction/formation of this disulfide is detected both by a shift on sodium dodecyl sulfate-polyacrylamide gel electrophoresis or by measuring the intrinsic tryptophan fluorescence of the protein. The six genes identified coding for Gpxs are expressed in various poplar organs, and two of them are localized in the chloroplast, with one colocalizing in mitochondria, suggesting a broad distribution of Gpxs in plant cells. The abundance of some Gpxs is modified in plants subjected to environmental constraints, generally increasing during fungal infection, water deficit, and metal stress, and decreasing during photooxidative stress, showing that Gpx proteins are involved in the response to both biotic and abiotic stress conditions.

The plant plastidial thioredoxins (Trx) are involved in the light-dependent regulation of many enzymatic activities, owing to their thiol-disulfide interchange activity. Three different types of plastidial Trx have been identified and characterized so far: the m-, f-, and x-types. Recently, a new putative plastidial type, the y-type, was found. In this work the two isoforms of Trx y encoded by the nuclear genome of Arabidopsis (Arabidopsis thaliana) were characterized. The plastidial targeting of Trx y has been established by the expression of a Trx::GFP fusion protein. Then both isoforms were produced as recombinant proteins in their putative mature forms and purified to characterize them by a biochemical approach. Their ability to activate two plastidial light-regulated enzymes, NADP-malate dehydrogenase (NADP-MDH) and fructose-1,6-bisphosphatase, was tested. Both Trx y were poor activators of fructose-1,6-bisphosphatase and NADP-MDH; however, a detailed study of the activation of NADP-MDH using site-directed mutants of its regulatory cysteines suggested that Trx y was able to reduce the less negative regulatory disulfide but not the more negative regulatory disulfide. This property probably results from the fact that Trx y has a less negative redox midpoint potential (2337 mV at pH 7.9) than thioredoxins f and m. The y-type Trxs were also the best substrate for the plastidial peroxiredoxin Q. Gene expression analysis showed that Trx y2 was mainly expressed in leaves and induced by light, whereas Trx y1 was mainly expressed in nonphotosynthetic organs, especially in seeds at a stage of major accumulation of storage lipids.Thioredoxins (Trx) are small (approximately 12 kD) ubiquitous proteins involved in thiol-disulfide exchange reactions (Buchanan, 1980;Holmgren, 1989). In higher plant cells, they are found in cytosol, mitochondria, and plastids (Schü rmann and Jacquot, 2000). All are nuclear encoded irrespective of their subcellular localization. The availability of the Arabidopsis (Arabidopsis thaliana) complete genome sequence revealed that higher plant Trxs belonged to a multigene family. Several different Trx types have been distinguished on the basis of sequence identity and intron positions, with several isoforms within each type (Meyer et al., 2002). The situation is particularly complex for plastidial Trx (cpTrx), where four different types of Trx have been found. Besides the long-time known thioredoxins m and f comprising 4 and 2 members respectively, a third type of cpTrx, named the x-type, has been recently studied and found to be a very good reductant of plastidial 2-Cys peroxiredoxins (Prxs), whereas having almost no activity with the classical target enzymes of cpTrx m and f, i.e. NADPmalate dehydrogenase (NADP-MDH) and Fru-1,6-bisphosphatase (FBPase). The fourth type has been identified quite recently (Lemaire et al., 2003) during the analysis of the genomes of Chlamydomonas reinhardtii and of the cyanobacterium Synechocystis. While only one isoform is present in the green alga and the cyanoba...

Thioredoxin f (TRXf) is a key factor in the redox regulation of chloroplastic carbon fixation enzymes, whereas glutathione is an important thiol buffer whose status is modulated by stress conditions. Here, we report specific glutathionylation of TRXf. A conserved cysteine is present in the TRXf primary sequence, in addition to its two active-site cysteines. The additional cysteine becomes glutathionylated when TRXf is exposed to oxidized glutathione or to reduced glutathione plus oxidants. No other chloroplastic TRX, from either Arabidopsis or Chlamydomonas, is glutathionylated under these conditions. Glutathionylation decreases the ability of TRXf to be reduced by ferredoxin-thioredoxin reductase and results in impaired light activation of target enzymes in a reconstituted thylakoid system. Although several mammalian proteins undergoing glutathionylation have already been identified, TRXf is among the first plant proteins found to undergo this posttranslational modification. This report suggests that a crosstalk between the TRX and glutathione systems mediates a previously uncharacterized form of redox signaling in plants in stress conditions. Chlamydomonas ͉ Arabidopsis ͉ Calvin cycle ͉ enzyme light-activation ͉ thiol T hioredoxins (TRXs) are small ubiquitous disulfide proteins with a conserved active site WC(G͞P)PC that play key roles in redox signaling by oxidoreduction of disulfide bridges of various target proteins involved in a wide variety of functions (1). In plants, proteins of the TRX family are found in most subcellular compartments, but chloroplastic TRXs have been extensively studied because they are key regulators of photosynthesis. Under illumination, the photosynthetic electron-transfer chain reduces ferredoxin (Fd), which transfers electrons to acceptors, including TRXs through Fd-TRX reductase (FTR). Four types of TRXs (f, m, x, and y) are present in the chloroplast with, generally, several isoforms for each type (2). TRXs f and m are involved in the regulation of key carbon-fixation enzymes that are mostly inactive in the dark and activated by TRXs under illumination (3). Some of these enzymes, such as fructose-1,6-bisphosphatase, strictly depend on TRXf. TRXf also appears to be the most efficient activator of other carbon-metabolism enzymes, with the exception of glucose-6-phosphate dehydrogenase. TRXs x and y, discovered more recently, are, rather, implicated in responses to oxidative stress because they are particularly efficient for the reduction of peroxiredoxins (4, 5). Moreover, recent studies suggest the existence of numerous other TRX targets for which the specificity toward TRX types remains to be determined (6-8).The thiol buffer glutathione (GSH) plays a key role in modulating plant stress responses (9-11), and work on animals has shown that GSH status can modulate protein activity through glutathionylation, a reversible posttranslational modification consisting of the formation of a mixed disulfide between the free thiol of a protein and GSH. This modification notably occurs in r...